How Plants Farm Microbes: Understanding the Rhizophagy Cycle

0:00 Yeah, thank you Noah, I appreciate that. Thanks everybody for joining us. We're grateful for you taking time out, and we know that there's going to be a large number of people watching this online later too, so we appreciate you taking the time to watch this.

0:15 We're really excited about tonight. Dr. James White is a professor of plant pathology and mycology at Rutgers University. And he's kind of taking the soil health world by storm a little bit, I think, because I know that up until a year or two ago I'd never heard of this guy, and now I see him all over the place because he's got this great talk about this rhizopg subject, which he's going to be talking about.

0:43 When I first heard him do this, it was on John Kamp's podcast, which we had John on last week. And by the way, Dr. White, John says hi and speaks very highly of your work. But when I heard you speak on John Kamp's podcast, I said, man, we've got to get this guy on board with some of the things that we're doing.

1:03 I reached out to you, I don't know when that was, six months ago or something. Asked you to write an article for a soil health resource guide, and you were gracious enough to provide that. So a little sneak preview for those of you: we will be coming out with a new version of that resource guide in January, and Dr. White will have an article in there on the same topic of rhizofiji and essentially how plants are essentially farming their own microbes. So it's a fascinating topic.

1:30 I will be the first to admit I do not understand it very well, so I'm looking forward to learning right along with everybody else. Dr. White, go ahead and take it away. And like Noah said, we will hopefully save 10-15 minutes at the end here for question and answer.

1:50 Okay, thank you Keith. Do you still see my screen or do I need to share again? I think you need to share again. I cannot see it right now. Okay, all right, that's fine.

2:03 Yeah, there you go. I can see you now. You got it. Okay, okay, okay, that's good. So I'm going to disappear. Okay, so Keith, you essentially said what I was going to do. You didn't steal all my thunder, but you introduced me very well.

2:21 I'm going to try to convince you that plants are cultivating or are farming microbes in their roots, and they are essentially tracking microbes to the roots. They're internalizing those microbes into the root cells. They're extracting nutrients from the microbes, and then they are ejecting surviving microbes back out into the soil to acquire more nutrients. So like we're farming plants in our agriculture, they are farming microbes in their microbial culture.

3:10 And in fact, these microbes really determine the nutritional status of crops, and they also determine the health of those crops. And these microbes happen to be endophytes. They live in the soil, but they're also taken to the plant. And these endophytes are microbes that go into the plant, and they don't cause any disease. Instead, they have beneficial effects in plants, and some of you may be familiar with the turf grass or the forage grass endophytes. These are fungi that go into plants, and in the turf grass industry, these fungal endophytes actually make the turf grasses heartier and resistant to disease and resistant to stress, and so forth.

4:02 This is a picture that shows one of these fungal endophytes in the grass. You see the cells in the background, you see the blue. That's the fungus. Okay, so these other endophytes that I'm talking about mostly are bacteria, but there are some fungi involved. But they'll go into the roots, and they're endophytes, and they have very similar effects.

4:23 So endophytes are really everywhere. Okay, all plants have endophytes. In fact, plants have really a community of endophytes that go into them. The roots will take in these microbes. They'll have multiple species of microbes that go into the roots. They'll also have endophytes in the leaves and seeds, but the ones that we're most interested in are the ones that go into the roots. And you really don't know those microbes are there unless you use a microscope and you use some special stains to visualize them.

4:56 So you can't. Okay, here's a plant that we just got legalized in New Jersey. We just legalized marijuana, of course we haven't seen any around here. But this is actually some seeds, marijuana seeds. If you look at these plants, if you germinate one of these seeds, they actually carry endophytes on those seeds. And those endophytes and the seeds will go into the seedling when it comes out of the seed, and it'll become an endophyte. Okay, and these are bacteria, but these also may be fungi. This is the hemp seed root. This is actually the root hair. You can see the picture on the left. That's the root hair. See the little brown structures in there? Those brown little dots in there, those are the bacteria. Those are the endophytic bacteria inside that root.

5:49 Hair and to the right you see that's another big hair and that is also filled with bacteria. You can see those spherical structures, those are all the bacteria there. Those bacteria are underneath the cell wall so they're inside that root hair cell but they're actually not in the membrane so they're not in the cytoplasm, they're just kind of underneath the cell wall.

6:13 So the those you would not be able to see those bacteria unless you use the special stain and you can see in the red written up the DAB, diamino benzodiazine stain that allows us to see those microbes. They're invisible without that and they we're only able to visualize them with that stain because the plant is secreting reactive oxygen onto them and then that DAB will stain that reactive oxygen.

6:41 This is another root here, that's another one of these hemp root hairs and you can see the bacteria at the tip and you can see little rods there, little blue rods there at the tip there and you see all the brown there that's where the bacteria are where the plant is secreting this reactive oxygen onto the micro. So the plant gets these microbes from two places. One place is on the seed so it's important that seeds carry the microbes with them so if you sterilize your seeds you kill these microbes and that's not good. And but also from the soil so there's two sources and the plant has to have these two sources in in order to get the best possible microbial community inside the plant so you have to have a healthy soil and you need healthy seeds that are not where the seed microbiomes have not been damaged.

7:38 So here's another plant, this is a giant cactus on a desert island of Bonaire in the Dutch Antilles and these cacti are all over the middle of that island, very huge plants and they have fruits way up there. If you take those, just like a prickly pear fruits at the little orange things you can barely see the dots here but if you take those off pull the seeds out it looks like this and then you take those seeds and germinate them it looks like this. And this actually, this is one of these cactus, Kadushi cactus they're called that has been stained with this DAB, this diameter benzene so we can see the bacteria. You can see the roots are orange because of that and if you look at that ceiling with the microscope at those roots here the root hairs, see the hairs coming across and you can see all these little dots in there. All those dots are the endophytic bacteria that came through that seed so this carried through the fruit, carried from the plant from the plant from the roots from the soil all the way up goes into the fruit and then it cycles through this plant. As this plant goes, this is an endophyte that this is one of these that's seed transmitted and the there the these seedlings are chock-a-block full of microbes.

8:56 This is a shows the hair again, root hair of that Kadushi cactus and you can see the little blue dots in there. Those blue dots you can see some of them are paired see where the arrows are, that's where they just divided okay. So these microbes are already in these plants they're inside the plant cells so that tells you how closely intertwined these microbes are to the to the plants. They actually become, you've heard of the microbiome, human microbiome we have microbes all over us well the same way with the plant the plant and microbes all over the all over the plant especially now in roots and root tissues and in the root cells in particular area around the root tips and i'll show you that. Turns out these microbes have all kinds of beneficial effects to plants and one is they improve the stress tolerance of plants. They make plants more resistant to stresses like heat and drought, heavy metals, other whatever is oxidative stress. So oxidative stress is really all stresses can convert into oxidative stress and so these endophytes protect the plant from oxidative stress.

10:05 They also suppress pathogenic fungi, suppress diseases so they protect the plant from diseases and i'll explain how that happens. They will control development or they'll modulate root development. Plants have to have these microbes in them in order to develop properly if you take away all the microbes they won't develop right they'll be sickly plants. If you take away all the microbes and you just give them fertilizer they will be weak plants they won't develop properly and they'll be weak. They'll grow big but they're going to be weak because they won't be stress tolerant and they won't develop quite right without those microbes.

10:40 The microbes in the roots also improve nutrient absorption. The microbes function in absorbing nutrients that's their main function in this cycle we call the rhizophage cycle and i'll explain that. That's the cycle where plants use microbes to get nutrients and finally the microbes in plants alter the chemical constituents of plants. If you have the right microbes in the plants the plants are going to have a particular chemical profile. They may have more antioxidants, that may have more chlorophylls, they may have more.

11:21 Keratins and so forth. If you put other endophytes in there, you don't have the endophytes, you'll change that chemical profile. It may not be as good. I mean, I don't know the particulars. It depends on the situation, but what microbes are involved.

11:38 In 2010, a group of investigators in Queensland, Australia discovered that plants were internalizing microbes into their roots. This young lady here, Channy Pongfoo, is one of the investigators that was involved in this Queensland Australia group. What they did was they labeled or stained some bacteria and some yeasts with green fluorescent protein, and then they fed them essentially to tomato plants and also to Arabidopsis plants. And then they visualized those roots of the plants with the microscope. With this green fluorescent protein, they could actually see where the microbes were inside the root cells.

12:32 They showed they went into the root cells. You can see, for example, image D here in the middle. All these little green dots, those are the microbes in there. They're actually inside the root hairs there, and so the root hairs are loaded. If you look at E down here, there's another one E down on the left. You can see a little hair there. You get little dots in there. That's a yeast in there.

12:55 What they also showed is that after three or four days the staining went away and they couldn't see the microbes anymore. Their concept was that the plants are actually eating these microbes, they're internalizing microbes and they're degrading microbes. They named this process rhizophagia. In a later paper, rhizo for root, phagia meaning eating. Rhizophagy. And this paper that they published in PLOS One was called 'Turning the Table: Plants Consume Microbes as a Source of Nutrients.' That was back in 2010.

13:34 We were looking at the process right around that time at plants degrading microbes on roots. But we actually at that time we hadn't looked inside the root. It was a little bit later, well after we saw this paper, that we began to look inside those roots. We discovered that in fact, not only were they degrading microbes on root surfaces, but they also appear to be degrading them inside the roots.

14:02 We discovered that it appears to be a cyclic process. What's happening here is that plants—this is a diagram of a plant root. Plants at the tip, right around the meristem, are actually secreting exudates. They're secreting sugars, they're secreting organic acids, they're secreting, in some cases, amino acids. So they're secreting nutrients outside the root, right around the meristem tip, right around the tip of the root. That attracts soil microbes to that root tip. They follow that nutrient trail. They follow right back to the plant, and the plant cultivates them there, keeps feeding them outside there. Then right at that zone where it's putting nutrients out, the plant internalizes some of those microbes into the root cells. It takes them in. We don't know exactly how it does that. It's just pure speculation what how it might be doing that. We just don't know the mechanism for the engulfing of the bacteria.

15:07 However, we don't know whether the bacterium inserts itself or it somehow gets pressed into the cells because the walls are thin there. So they could easily penetrate into those walls. But we do know they go into the walls because we can see them there. If you look at the picture to the right, you see at B. You see the arrow there at B pointing. Those are bacteria inside those cells, right around that area where they're entering. That's the epidermal cells, the root cells where they go in.

15:39 Well, what happens is once they go in, the plant hits them with reactive oxygen, superoxide. That knocks their cell walls off of the bacteria or the yeast. They form these protoplasts. The microbes are naked protoplasts. They continue to hit those protoplasts with superoxide, which makes those protoplasts leak. Nutrients are leaking. Then those microbes—some of those microbes degrade, but others of those microbes survive. The survivors will actually trigger root hairs to form on the roots.

16:17 If we don't have any microbes on the roots in the root cells, they do not make root hairs. So we can deprive seedlings of root hairs by removing all the microbes. We can do that by very rigorous sterilization. We can remove all the microbes. But if the microbes are there, they trigger root hair elongation. Then as those hairs elongate, those microbes are shot out into the soil through little pores that happen in the dip. They go back to the soil. Once they go back out into the soil, they reform their cell walls. The microbes do. And actually, the plant gives them nutrients—enough to reform the cell walls because what happens is it takes.

17:01 It shoots out the tips of the hairs some other exudates that enable those microbes to reform their walls. Then they'll reform their walls if they have any swimming structures like flagella they can reform those and then swim out to the soil and get more nutrients.

17:16 Over here at C you can see the actually a root hair tip and this is a fluorescent stain but you can see all the microbes that are coming out of that hair tip. And I'll show you actually some movies that show that actual process because that's a still with fluorescent microscopy.

17:34 So here's a seed a grass seed germinating and you can actually see the root down here at the bottom the lower arrow and you can see where the shoot is coming there. And you see some yellow around there that's bacteria that are coming off.

17:49 So these actually these seeds will carry microbes with them a grass seed will carry you know a half a dozen or so different or more different microbes that will then colonize that seedling when it germinates and go into the root and also go into the chute. It's just that we you know the rhizobium cycle doesn't involve those microbes on the chute but they're also there.

18:15 But they are on the root and they'll colonize that root and the root actually colonizes them instead of the or will feed them and what it does is this shows a diagram here of a root and you can see this little hump coming out here that says root exudates. That actually is where the root is secreting those those sugars and and organic acids and amino acids to cultivate those microbes and attract those microbes.

18:45 So all roots do that and all roots that produce root hairs will do the rhizophagic cycle. So the right the root hairs appear to be directly connected with rhizomehagia cycle we always think that root hairs since third grade you know we all learned all of us every bit of one of us I think I did learned that root hairs function in absorption and that's the role of root hairs. Well with the rhizophagia cycle it appears they have a different function and that function is to eject those microbes back out into the soil. Elongate it out of the soil into the rhizosphere where they can put those microbes out where they can get some more nutrients and then bring it back to the plants.

19:28 So this shows a root tip and you can see the the the dense blue area there at the tip that dense blue area is where the microbes are entering. So the they're also staining better there with this blue stain. This animal and blue stain and you can see this cloud of blue dots around it around the outside those are all microbes out there there's a cloud of microbes that the plant is colonizing is is cultivating right out there. And then taking them into the cells if we look closely at these cells you'd see they were getting filled with these microbes with these bacteria going in.

20:06 Once the bacteria go in the plant will squirt superoxide reactive oxygen I know I keep saying reactive oxygen superoxide which is a highly potent form of reactive reactive oxygen and the superoxide gets produced by plants on the root membrane the root cell plasma membrane. So the membrane on the root cell it actually can detect those microbes out there we don't know how it does it but it appears to detect the microbe and then it squirts superoxide on it and it squirts superoxide from this kind of a it's an enzyme NADPH oxidase or NOX just knocks enzymes in the and that enzyme will take oxygen and produce superoxide. That superoxide you see there in red that's the potent molecule.

20:53 And as it turns out the the superoxide is also a defensive molecule so it's something that plants will do to defend themselves okay and that doesn't mean that this is a microbe attacking it it means in this case it is a plant that uses this same molecules superoxide to extract nutrients from those microbes. So this superoxide will strip the cell walls off oxidize the cell walls right off of those of those bacteria and this is what it is what it looks like okay. This is the bacteria when they when they have their cell walls to the left and you see their rods okay that's with cell walls once they lose their cell walls you see these little spherical things they no longer have rod shapes they become little spherical.

21:42 And they can just bud real fast and so once the once the plant once the root cell removes the cell walls off those microbes and forms these little protoplasts then it can replicate them also real fast it can also break them down very fast so a lot of these get broken down but others can be replicated to smaller and smaller many more essentially they can be cloned. So those that survive this oxidation process they can be cloned and and then replicated so the plant actually makes more of them. So a few will go in and many more actually are ejected back out into the soil so the plant is cloning these microbes okay. This is a cell wall this is bacterial cell wall what's the significance of the taking the bacterial cell wall off well one significance is there's protein in those cell walls so that's nitrogen so the plant actually can get

22:38 Nitrogen by degrading the cell walls off of those microbes and these peptidoglycan protein you can see protein in both of these peptidoglycan. Those are all proteins so that means the plant is getting nitrogen from taking those cell walls off.

22:52 This is what one of these root cells look like with these microbes in it. And you can see they're cylindrical kind of shape and then you see these little spherical structures that are staining red. Those are the protoplasts of the microbe that have been taken into that root cell. And you can see there to the lower arrow you can see there's a chain of three there and you see that there's a little one there's dark blue. That dark blue, the blue is protein and the lighter ones have more protein removed or less protein so they're breaking down. So they're being replicated but some of those are breaking down and the microbe and the plant is getting nutrients from them.

23:35 So when those microbes go into the root cell they actually don't go into the cell itself they just go underneath the cell wall. This is a diagram you can see the gray here this is the cell wall and you can see the blue space underneath that cell wall is a space called the periplasmic space or it's just a space under the cell wall but it's not quite inside the cell. The cell is this white and so what's happening here is the plant is keeping the microbes outside that cell but something else is going on and I'll show you that. It's also besides hitting them you see the 2O the 2O minus there. Those are the superoxide but also the plant is turning those microbes around and around. It's churning them in something called cyclosis. It moves them around and moves them around. We think that's important because that will break up the microbes in the smaller and smaller pieces, smaller and smaller cells but it also will break down any gradients that form in nutrient exchange.

24:43 If the bacterium stays still then nutrients flow from the bacterium to the plant and a gradient forms. A gradient means it's concentrated in one area and less and less concentrated but that slows the transfer. If they're moving like that the gradients break down and the transfer is fast. So what that enables is maximum transfer of nutrients between the microbes and the root cells. So that cyclosis, that's called cyclosis.

25:09 Here it actually shows a root hair and you can see it's stained for superoxide. This purple stain something called NBT stain but it stains superoxide it stains blue. And you can see it blue purple you can see those little blue dots all around. You can see at the tip there all that blue in there that's where all the superoxide is concentrating at the root hair tip but you see it also little blue structures all along so those microbes and the root hair tips are really being hit with superoxide.

25:44 The microbes in the inside these roots trigger development in two ways. They'll essentially if you remove the microbes you have low development. What happens is roots no longer. If you remove all the microbes if we sterilize the seeds so we remove the microbes and seedlings will produce roots where the roots will not have the gravitropic response. Normally roots will grow down like this go down to the soil that's with the microbes. You take the microbes and they lay flat on the surface or they go into the air. They don't, they no longer they no longer have that gravitropic response. The other thing that happens is they no longer. If you remove the microbes they no longer form root hair so you have no root hairs forming. So those microbes are really important for development of those of those plants seedlings. They need them there. They're part of development.

26:37 This shows a root of a grass, this one bermuda grass seedling root and you can see the root tip over here the left one. And you see the tip over there the cells going off those cells falling off those are the root cap cells. You see a little bit older area to the right and you see there there's no hairs there's no hairs or if you do you see little short things that haven't elongated. These are without microbes. Without microbes you get no hairs. In the same experiment we take a microbe and then put it on that. We removed all the microbes then we take it and put it on and then what happens once we put the microbe on you see this now. Now you see the tip you see the root hairs you see the blue hair is forming they begin to form right away. You also see this is darker. There's more reactive oxygen here with these microbes there. This reactive oxygen relationship that interaction between the microbe and the plant causes this brown staining now. So this you see that now. If you and that has a big effect all over the plant in fact but the point here is that with those microbes you get proper root development. Without them plants they don't develop right they're sick.

28:00 Can see the white arrows that I'm showing. The lower white arrow there, kind of see this brown elongate thing kind of divided into two. You can see a line in the middle. That's one of these bacterial rods or two of them actually of those rods. You can see some others in there, some rod shapes structures, brown structures. Those are the bacteria with their cell walls.

28:20 So they have just now entered into these cells. They have their cell walls and the plant is beginning to hit them with superoxide that will knock those cell walls off, but you still see them at this point. They're still there. So initially when they go in, they still have their cell walls and that's how we know that they went, how they, how we know they went in here because we could see them here. But we can also see once they go in, they still have their cell walls and shortly after this they will lose their cell walls and then we track them through the plant.

28:50 This actually shows a root, a more mature part of a root. And this is with the bacteria here. This is like a pseudomonas endophyte in bermuda grass and you can see these blue hairs. You see all the little brown dots. Those are all the bacteria inside those hairs. And if you look at the closely at the root body itself, you can see little dots all over it. Those are all the bacteria inside those roots, inside those plant roots. They're inside.

29:16 The plant has taken them inside and you see here's a root hair and you can see the little red dots, brown dots. Those are all the bacteria protoplasts now inside that hair. Inside the root here they become filled. They become engorged on these bacteria. And these, I should say these, you cannot see unless you use a special stain to visualize them. A lot of old illustrations of root hairs have shown root hairs and they just show little, the little bubbles in their little spherical things and they've always called them bicycles. They didn't know they were bacteria in the hairs, in the in the old studies.

29:59 So it's easy not to see these. You have to really, you have to really look for them and you got to use a special stain to see them. Okay, so this shows a root, to the left and you can see the root tip there. You can see a little cloud of microbes around that root tip in the dark area and you can see the hairs. The hairs of course are where they're ejected back out into the soil.

30:20 And if you look at the middle one you can see the hairs there. This is a fluorescent stain. You can see all these dots, these fluorescent dots of green dots. Those are the bacteria coming out of those hair tips. They're ejected. It's like, it's like, you know, these hairs will eject periodically squirt those microbes out and then they'll grow a little bit and squirt more microbes out and grow a little bit and squirt more microbes out, so they do this periodically.

30:45 Okay, you look to the right you can see there. You can see a hair, another kind of grass, and you can see the bacteria coming out of the tip there, coming out of that hair.

30:57 So we actually developed a, well it's a hypothesis for how it works but it's a model of how ejection of microbes is happening from these root hairs and we call it the cyclosis expansion wave mechanism for microbe ejection. And that is, this shows, over here at one, image one to the left you can see this is a diagram of a root hair and you can see these black dots there. Those are the bacteria actually in the and the solid line that's the cell wall of the of the root hair. That's the root hair wall.

31:39 And if you look at the dotted line inside, that's the, that's actually the membrane of the root hair. If you notice that membrane of the root hair, it is like a needle shape. It's a pointed, pointed shape. It's a pointy shape. It doesn't go all the way to the edge of the of the of the wall. Instead it's skinny, a little skinny hair in the middle and instead what you have are these microbes that fill in the rest of that space. And they accumulate out there.

32:11 And what's happening is the there's what we call cyclosis. That is the root hair is moving those microbes around. It's moving cytoplasm around. It's moving the microbes around in a circle counterclockwise or clockwise either way it goes. It moves the microbes around. Since tends to go in one direction maybe it'll reverse at some point but we've only seen it going one direction at a time. And then it moves those microbes to the tip.

32:40 They accumulate out at that tip. They accumulate at the tip because the cell wall here is very elastic and so it'll swell. It will, it's elastic, it'll move but along the lateral walls the cell wall is thick and hard and rigid. And so they move to the tip and then they'll, then they're not easily moved from the from the tip back but occasionally some will be caught and moved around. Okay, so that keeps happening like that and then what will happen is there becomes an expansion wave that begins at the base of the hair.

33:15 And then it expands all the way from the base going out to the tip and as it expands it will push those microbes. You can see the microbes, they'll expand like a bubble, push those microbes out the pores that happen in the tip and it turns out those microbes are protoplasts so they can go out very very tiny holes, very tiny pores.

33:38 So once they're out there they'll reform their cell walls and then go back to the soil but I'll show you more of this. So what is that expansion wave? I think I'll discuss that in a minute. This is a sedge that occurs in the rocks and on that desert island of Bonaireone of the nice things about my work is I get to go different places and so it was nice to do that research on that desert island. It's a benefit of doing the work that I do on plants, studying different plants.

34:08 So that was a sedge that occurs in rocks and this is the root hair of that sedge. And what the root hairs lack is they don't have a lot of soil on those rocks but they do have microbes. This shows the root hair on the bottom and this is stained with that reactive oxygen stain so you can see the microbes and you can see the arrows there indicating where the red is. There's a blue microbe in the middle, maybe one or two in the middle there, and you see another one, another arrow there to the right, and you can see there the red there, but you also see the blue. That's where the microbe is, it's out there.

34:40 And now this is the living hair and this is actually a movie we made. We grew these things, these sedges on in an auger, this gelatin, and then we visualized it from the reverse of a petri dish which is plastic, clear plastic, so that we could see the hairs, actually the microbes moving in the hairs.

35:08 And what you can see here is that cyclosis is happening. You see these microbes are moved, looks like counterclockwise here. You can see where the shadows are, those are where the microbes are and they're continually moved around like that. And as I mentioned, the importance of that movement is it breaks the microbes up, it clones them so you get a lot from just a little, but it also speeds up the transfer of nutrients from the microbe to the plant so the plant can get more nutrients from the microbe. So it breaks down those gradients.

35:40 This is a hair from that sedge and you can see what we're seeing here is clusters of those little cells. Some of those are big and some are little. That probably came from each cluster. Probably came from one microbial cell that was broken up into many little pieces and you see all the little ones and the bigger ones there. So that's what's happening. The plant is cloning these microbes. After it extracts nutrients, it's cloning them and it's going to push them back to the soil to repopulate the soil with these microbes.

36:12 Okay, so this is that hair again. This just shows the tip though. And here you can see the hair. You can see the microbe going along the shadows, going along to the tip and you see them. Now you see the little shadows, the little spherical structures bouncing around the tip and that's what happens. That's how they look right there. And so this happens and they accumulate there and every now and then you may see one that gets caught and carried back again but this is they stay out here.

36:43 And in staying out here they actually can control development of that hair and we'll talk about that or they'll have, they'll play a role in development of that hair.

36:55 Okay, this shows that ejection. This is a picture of that. This is the hair again, that's sedge and you can see all the little red dots in there. Those are all the micro protoplasts inside and you can see how fat it is. The hair, the actual hair is a little skinny little thing in there. All of this fatness out here is microbes. These microbes are, these hairs are engorged of microbes. These hairs are, it's incredible how many microbes get in there and they eject them out the pores, the thin pores here at the surface pores that form at the tip and you can see here around here where the black arrow is. This is where the microbes have been ejected and they're reforming their cell walls here. Once they're ejected they're no longer exposed to superoxide. They're reforming the cell walls. They go back to the soil.

37:49 Okay, so this actually shows this young lady to the left, Sophia Davinski, actually did the work on this that helped to generate this movie. But this is tomato and it's showing the actual ejection process. And if you look over here at one, over here all the way to the left you can see that expansion starts at the base. You see like a bubble that goes up and if you look at the middle one you can see it pushing, pushing, and as it does it pushes microbes out. To the one all the way to the right, number three, they go out. And once they're pushed out, now what we think is going on, we have some data that this is probably potassium loading, that the plant is putting potassium into the vacuole in this hair and causing water then to go in and so then it rushes through out the hair and pushes them out but we have yet to prove that.

44:28 All the microbes, and then we put it back on the one to the left, E plus, that's endophyte plus. This is rice. You can see those seedlings are big, and we left it off of the one to the right, E minus, there. And they're little tiny. A little bit later, you can see the seedlings, it's the same scale, E plus to the left. You see how big it's—like double the size of shoot. The root, maybe the root is the same length, but look at the soil. The soil adheres to this root with the endophyte. Over here to the right, no endophyte, there's very little soil. If you look closely at those roots to the right also without endophyte, there's no root hairs, or very few root hairs that have formed.

45:10 So the whole relationship when you have the microbes there, the whole relationship with the soil changes. The plant with those microbes there can now get those nutrients out of that soil. It's got a closer relationship to the soil. It's a major difference in terms of nutrient acquisition, for one thing: development and nutrient acquisition.

45:32 Okay, the nutrient function. Okay, we've been working on this of the rhizophagia cycle, nutrient function of the rhizobium cycle. This is an experiment done by a visiting scientist, a young professor from China who came over for a year in the lab. One experiment that he did—we've done several of these experiments now—but I'll just describe this one. He basically took wheat, removed the microbes, and then he put the microbes back on the wheat one at a time, planted in potting mix, and grew them.

46:06 Okay, so this shows actually no bacterial—this is just control, all bacteria removed. And you can see these are very tiny roots, stunted roots and shoots. And then he put one of the microbes, the Bacillus, back on, and you can see big roots like three, four times the size. And you can see shoots like one and a half or double the size. It made a huge difference in growth of the plant. And that's what you can see nutrients are. If you look at nitrogen, okay, without bacteria, you look to number one over here to the left, you can see it's so much nitrogen here, this blue bar. And then you put these bacteria back on—he had three bacteria that he put back on—but in terms of nitrogen, they all seem to be doing about the same. But they—the plant benefited in terms of nitrogen. If you look at phosphorus, same deal. Okay, phosphorus, no bacteria was down here. They all benefited with phosphorus. Potassium looks like number two over here or strain LB1 over here. That actually did extremely much better. It looks like three times where the no bacteria were, and the others—the other bacteria—did double. So I mean already you can tell it does matter what the bacterium is in terms of what nutrients plants get.

47:26 So certain microbes will carry certain nutrients, other microbes, other nutrients. And so they're not all microbes aren't equal. So that's an important something to remember. Calcium, this is calcium as you know, back here, and then you see with the bacteria, you see a difference. You see an increase. Here's sulfur, no bacteria, then you see an increase with sulfur. And manganese, okay, none, very little, and then almost double with manganese. Magnesium, okay, 30 more. Zinc, okay, 20 or 30 more with the microbes.

48:08 Okay, so this is a point I already made, and you didn't see it very much with in this experiment, but in other experiments we've done, we can show major, major differences depending on which microbe you use in terms of which nutrient the plants get.

48:26 So can we add commercial biostimulants to crops? Okay, that was a question: are they functioning in the rhizophagy cycle? What do they do? How do they function in here? So Ivy again, you know, did this. It was important because it's an industrial project, so we're interested in what's happening in the industry out here. And when you know, will their microbes work also? And how will they work? And so she's looking at some of that for her PhD.

48:52 And she put some commercial microbes in celery. She cleaned the celery up, and then she took those commercial microbes, made sure they were clean, and then put them into celery. And most of these commercial, most of the ones we looked at were Bacillus. And they go in as endophytes. They're not all equal. There are some again that are better than others, but they all appear to be endophytic. And at least the ones we looked at—we didn't look at a great number of them—but this is actually the control with no microbes. You can see there's no hair here. This is the root tip. This is celery. And you can see with the microbe, this is Bacillus amyloid liquifications, one of the biostimulant product microbes. And you can see root hairs forming there where these microbes went in and they became endophytic and stimulated hair formation.

49:50 You can see there's a close-up of the hair with the microbes in it. You can see the hair there. You can see the little brown in there—those are all the microbes there—and the microbes coming out on the surface too. You can see them there. You can see. So these commercial strains do work. You always need if you use commercial.

50:07 Strains you always need to check and make sure they're working on your crop. You want to make sure no matter what product you get, you want to test it as much as you can yourself.

50:18 Okay, here are microbes. Microbes also alter the chemical constituents of plants, and this is an example here in the tomato experiment—carotenes or carotenoids. And had a student that took some microbes off of some tomato or some carrot relatives and then put them on carrot and then measure the carotenes in those carrots to see if any of them increased carotene formation, and she found three bacteria. Back I'll just say bacterium one, bacterium two, and bacterium three. And then she put them, and I should say one is from celery, one is from cumin, one is from parsley. And then she put them onto carrot, and you can see these microbes and the carrot hairs there. You see little dots there. You can see them inside the carrot root cells there. There's little clusters, a little spherical cluster. Those are all the microbes in there.

51:17 And here's then what she did is she analyzed the carotenes in the carrots themselves. And this shows actually alpha carotenes, and you can see the control over here looks like it's all the way to the left. Control you can see it's a little bar. Then you see bacterium one. Bacterium one didn't do anything in terms of carotene formation. It was not effective. Bacterium two looked like it almost tripled the carotene content alpha carotene content, and bacterium three looked like it increased it slightly.

51:52 Okay, here is beta-carotenes. Okay, exactly the same way. You see bacterium two—it made a major increase. Bacterium one almost did nothing. You wonder if bacterium one is actually colonizing the root. It may not even be colonizing. Okay, bacterium three increased it but not as much as bacterium two.

52:16 So the chemical components, the antioxidants for example, the pigments and other components that people might consider, they might consider them stress tolerance molecules for the plant, or they might consider them health components. In the case of carotenes, or we know also luteins, some other kinds of pigments we also looked at that are also increased in plants with certain microbes. So those, you know, you can't alter, alter that. Improve the quality of plants, improve the quality of crops using microbes. We don't know all the details how, and every plant may be different, and different microbes have to be experimented with, but there's something there.

53:08 Okay, so this is a diagram that just shows how the relationship of these microbes is affecting plants, and how if you look at number three here you see a plant model, and the plant basically, going into the soil and cultivating these microbes, is in essence partaking or participating in the microbial community. It's getting those microbes, it's cultivating those microbes, it's getting nutrients from those microbes, and it's giving nutrients to those microbes. Those exudates are nutrients, and it's taking those nutrients. So there's a flow back and forth. The plant, in essence, becomes a part of that microbial community.

53:51 There are up at a you see this little square, three beneficial outcomes of rhizospheric symbiosis or rhizospheric cycle. And that is one: the plants get nutrients from the microbes. That's critical outcome. But also because these microbes are going in, at two here, because the microbes are going in and the plant is interacting with them oxidatively with superoxide and other kinds of forms of reactive oxygen, the plant has to upregulate its own oxidative stress genes, its own antioxidants, its own protection from the superoxide that it's producing. So it does that, and then the plants become more resistant to oxidative stress, more resistant to stresses like heat, drought, heavy metals in the soil, salt tolerance. All that converts to oxidative stress, and these plants are already resistant to oxidative stress, so they can resist all that. That makes plants heartier, and they're more resistant to climate change and more resistant to whatever happens, to drought that might happen, maybe lack of oxygen. Even gives some oxidative stress, lack of. Believe it or not, flooding for example will affect plants, and they could also be more resistant after that.

55:04 So and but there's another effect, and that is the microbes will also go out of the plant and they will colonize pathogenic fungi, any fungus really, in the soil. They'll colonize that fungus. We've seen a lot of these do that. Bacillus species also pseudomonas. They'll go out and they go on the surface of these fungi all over the hyphae, and they will cause them to leak nutrients. The fungus will then leak nutrients. The microbe could get it and carry it back to the plant. Okay, but the fungus leaks those nutrients, and it no longer has the energy to cause disease, and those fungi then become. We can see the whole behavior.

55:46 Change they no longer sporulate they'll grow without sporulating. They'll grow slower they'll colonize the plant the pathogen can colonize the plant but it's not virulent. It'll grow on the plant these microbes will be all around these bacteria will be all around it so it won't cause disease. In some cases we've seen for example in fusarium with the bacteria present the fusarium becomes an endophyte in the plant it's emphatic it's not pathogenic.

56:12 So these microbes that are involved with the plant that the plant is cultivating protect it in many several different ways. The whole health of the plant is improved development health and so forth.

56:26 So my very interest in concluding my take homes here are that seeds should not be sterilized you should preserve microbes on seeds seeds as much as possible. You should put microbes on seeds. We've lost microbes in a lot of our seeds so experimenting with putting microbes on using products is a good idea that doesn't mean put it on all your crop but you should do some trials with some of these seed treatments to see how they're working.

57:00 Try not to use seeds where they've been where the husks have been removed a lot of seeds have seeds husks removed cotton is treated with acid that's a problem I don't have a solution to that but the acid kills all the microbes on the seeds and leaves the cotton at a disadvantage and to pathogens and everything else.

57:23 So you know but anyway seeds with their microbes they're better manage soils so that you build up the microbial community. Okay and you can use biostimulant microbes. They do work they may not work in all circumstances and this is the case in my view this is a case where you should do a trial and see how they work in your in your field and your crops with the plants you're planning.

57:54 So these are some references rhizophagescycle we published a couple of articles about this in 2018 and 2019. Those are the major articles and those are open access you can get to those. I'm happy to answer any questions about any of this and these are all the people who could contribute to this over this number of years. We've been working on this a long time and so a lot of people all these people have had their little contributions to that so thank you very much.

58:24 Keith I see you ready for yeah I know that was great Dr. White we sure appreciate that that's such a fascinating topic. I think we could spend a long time with questions but I do want to get to a few questions that people have submitted. Yes I could ask you questions for a couple hours myself but I'll share the time here.

58:44 There were several people that asked the question you know with this whole process how do you know like neonicotinoid seed treatments or other type of fungal seed treatments and things like that does that affect this does that harm the microbes and affect this rise of phagy cycle.

59:02 Well rhizophagia cycle is mostly bacteria but there are fungi that go in and it depends on the plant. Some plants will have yeasts that go into them and but a lot of those are like amaranth for example amaranth will have black black yeast that will go into the cells into the root cells internally colonize the root cells and I would think if you were using you know I don't think anyone uses that those fungicides on amaranth this is mostly a like a specialty crop I don't think they do anyways.

59:40 Generally I think neonicotinoids should only affect the fungi and bacteria should not be impacted so I would think that would have that would not have a major impact but you know we haven't checked it and it's certainly entirely possible and we just haven't gotten that far into testing you know fungicides. So in other words anything that's going to hurt that microbial community on the seed yeah we don't know we don't know all the effects exactly. Yeah it's a bad idea in general it's a bad idea to treat seeds with noxious chemicals.

1:00:29 Now I will tell you I will tell you many of our seeds and this is highly relevant here many of our seeds you know just in cultivating those crops have lost beneficial microbes that were present in in nature. I mean there was a study that some people did with the wild tobacco where they cultivated this wild tobacco for like seven years and after seven years they every year they would take it and bring it in and store it and plant it again it was an annual and they did that seven years and then it started having a major disease and they went back and they looked it was a wilt disease that happened.

1:01:12 They went back and they looked at the wild plants and they found they had microbes there that were preventing the wilt disease then they went back they got the microbe they put it back on their plant and they cured their disease. So just the nature in that experiment just the nature of taking plants bringing them into cultivation causes them to lose microbes so I mean we may have lost in many of our crops not just things

1:01:38 Like cotton where we acid treat them, but many other crops lost critical microbes that were protecting those plants in nature, and we may have to put them on. In some cases, using fungicides may be the way that we are compensating, right? By putting those fungicides on.

1:02:00 As a seed company, I've always wondered about this question. Do you feel like if we're selling seeds that were grown in kind of a regenerative soil health based system, is that seed going to be better than something grown in conventional ground? And then the second question is a lot of the cover crops that we use are old varieties, you know, like iron and clay cow peas, you know, 100 year old variety, really haven't had any breeding done on them for a century. Do you think those are better seeds than something that's been bred for high yield, high seed production yields recently?

1:02:39 Good question. First, the answer to the first part: yes, regenerative soil, the biodynamic conditions, right, biodynamic conditions are better because the seeds that can acquire microbes in the process, if they're grown properly. And in terms of the breeding, the issue with modern breeding programs, there are two issues. One is in some cases, like for example corn, they go through tissue culture, which sterilizes everything. The other issue is they're actually selected for response to nitrogen, right? But they'll respond to nitrogen and other fertilizers, and that might be doing something to the plant. This doesn't happen in nature. In nature, plants are doing rhizophagy cycles. That's what they all do in nature, that's how they do it, that's how they get their nutrients. They're doing this and they're absorbing, you know, other things. When you give them nitrogen, they don't have to work for it, and they're going to show they're going to be weaker, lazy, and they're not going to be oxidatively resistant because they don't have to break down the microbes oxidatively. You're giving them nitrogen, they get everything they need with that, and so they're not going to be hearty.

1:04:14 We've got a question here asking: can compost tea or compost extract work to inoculate seeds or the soil? And since we don't know exactly what microbes those plants use, something like a compost is just going to have a wide spectrum. Is that going to be helpful or useful?

1:04:33 Yeah, I think it would be. But like anything, like the commercial products, you know, I'm a big advocate that you should experiment with it. And I mean, I would expect, in fact my expectation is that if you can get a diverse community into your soil that might not have it, you'd be better off than if you left, say, a depopulated soil without a microbial community. So something like a compost tea would be a good idea, something worth doing. But you should experiment with it. I mean, farmers and growers, all kinds of growers, are adept at testing products on a little piece of land. So probably the more beat up and worn out that soil is, the more some of those treatments or amendments might help. But if I'm coming into a really rich, really regeneratively grown soil for many years, and it's already got a full biological profile, I may not see much of an effect.

1:05:45 Well, Dr. White, I would love to spend a lot more time asking questions, but I don't want to be sensitive to everybody's time. We've got a lot of people, and we need to let them get on to their next thing. We're very grateful. I would encourage everybody to share this. Noah will email out a link for the recording when we're done here in the next few days. If you've got people that you think need to watch this, certainly get this to them. Again, Dr. White has written an article that we'll have under Next Soil Health resource guide. So we're excited about that too. That kind of sums up a lot of this, but so much great information, Dr. White. We're very grateful for you sharing your time and your knowledge and expertise with us this evening.

1:06:28 Noah, do you have any comments about what's coming up next?

1:06:33 Yeah, first of all, thank you, Mark White. That was very good. Learned a lot. As Keith said, I will be recording this, so we will be getting this recording out here in the next couple days. And as far as any questions, you can just relay them back to us. If you did not get them answered, and we can try to get those answered for you or direct them to Dr. White.

1:06:54 As far as next week, we do have Keith is going to be interviewing Trace Genomics, and that's going to be John Jansen, I believe, so we're really excited about that and the opportunities there. It's not necessarily the same topic, but something along the same lines as far as testing the genetics of seed and really looking at what those plants are doing for the soil. So we're going to kind of continue on the same trend here next week. But thank you guys so much for watching. Thank you, Keith, and thank you, James, for your time. We really appreciate it.

1:07:26 My pleasure. Thank you, thank you both. Thank you. Good night. Have a great evening.